Summary

Embryonic stem (ES) cell lines have provided very useful models to analyse
differentiation processes. We present here the development of a
differentiation system using ES-like cell lines from medaka. These cells were
transfected with the melanocyte specific isoform of the microphtalmia-related
transcription factor (Mitf). Mitf is a basic helix-loop-helix-leucine zipper
transcription factor whose M isoform is restricted to neural crest derived
melanocytes and is essential for the development of these cells in vertebrates
from mammals to fish. What is not clear yet is whether Mitf is a downstream
factor or a master regulator of melanocyte commitment and differentiation.
Expression of Mitf in the ES-like cells from medaka led to the induction of
cells that, by morphology, physiology and gene expression pattern, were
confirmed to be fully differentiated pigment cells. Mitf expression is
therefore sufficient for the proper differentiation of medaka pluripotent stem
cells into melanocytes.

Introduction

The mechanisms that regulate the decision of a pluripotent cell to
differentiate and which lineages will be followed once differentiation is
initiated are intensively studied. Embryonic stem (ES) cell lines,
undifferentiated cell cultures that retain their pluripotency after long-term
cultivation in vitro, have provided very useful models to study how these
processes take place in vivo (Donovan and
Gearhart, 2001).

However, although most results provided some indication that a variety of
developmental decisions can be modelled using ES cells, attempts to direct ES
cell differentiation in a homogeneous fashion are still in their infancy. In
general, the methodology for inducing differentiation in mouse ES and EC cell
lines involves formation of embryoid bodies, cellular aggregates that undergo
a program of differentiation reminiscent of early mammalian embryogenesis
(Robertson, 1987). The
limitations of the embryoid bodies affect the general applicability of this
approach, particularly the activation of inappropriate signalling pathways as
a result of the inherent disorganisation of these structures
(Rathjen and Rathjen, 2001).
The development of other strategies that can promote differentiation only
towards specific directions is necessary and will allow the controlled
reconstruction of entire single lineages in vitro from pluripotent cells to
functional, terminally differentiated progeny.

We have developed a differentiation system using ES-like cell lines (MES)
from medaka (Hong and Schartl,
1996; Hong et al.,
1998). These cells show similar characteristics to mouse ES cells
in vitro and in vivo. Upon certain rather non-specific stimuli, such as high-
or low-density culture, embryoid body aggregation or growth factor treatment,
they can differentiate in vitro into many different cell types
(Hong et al., 1996), including
melanocytes, neurons and myotubes. After transplantation to host embryos, MES
cells participate in chimera development where they contribute as functional
cells to the formation of tissues from all three germ layers
(Hong et al., 1998).

Melanocytes originate as non-pigmented precursors, termed melanoblasts,
from the neural crest. After migration and proliferation, they enter the
epidermis and are found as mature pigmented cells in the skin as well as a
range of other sites where their role as pigment-producing cells is less
understood. The large amount of functional genetic data already available from
this lineage has made the study of melanocyte development an attractive system
for understanding how the integration of gene expression and signal
transduction pathways governs the development of a specific cell lineage
(reviewed by Goding, 2000).
Various factors, including colony stimulating factor (SCF), endothelin 3,
12-O-tetradecanoyl-phorbol-13 acetate (TPA) and cholera-toxin have been shown
to stimulate the differentiation of melanocyte precursors
(Murphy et al., 1992;
Ito et al., 1993;
Ono et al., 1998). Some
melanocytes were obtained after seeding undifferentiated ES cells on a bone
marrow-derived stromal cell line and treating the cells with dexamethasone
(Yamane et al., 1999). Mature
melanocytes were obtained from a cell line of immature melanocytes treating
the cells with retinoic acid (Watabe et
al., 2002). An increase of expression of microphtalmia
transcription factor (Mitf) was observed indicating an important role in this
process. Moreover, expression of Mitf in fibroblasts conferred some
characteristics of melanocytes on a part of the recipient cells, like
tyrosinase expression and dendritic morphology
(Tachibana et al., 1996).
Similarly, ectopic expression of Mitf induced transdifferentiation of
neuroretina into melanocytes (Planque et
al., 1999), and overexpression of Mitf in zebrafish embryos
resulted in ectopic melanised cells
(Lister et al., 1999).

Mitf is a basic helix-loop-helix leucine zipper transcription factor that
is expressed in multiple isoforms. Expression of Mitf-M is restricted to
neural crest derived melanocytes and is essential for the development of these
cells in mammals and in fish. Mice bearing null alleles of the gene exhibit a
loss of neural crest-derived melanocytes, associated deafness and a failure of
retinal pigmented epithelial cells. In zebrafish, a null mutation results in
loss of dermal melanophores but does not affect the retinal cells
(Moore, 1995;
Yajima et al., 1999;
Lister et al., 1999). It has
been shown that Mitf regulates the expression of the tyrosinase, Trp1
and Dct genes, the enzymes that produce the pigment melanin (for a
review, see Goding, 2000).
However, because Mitf is essential for the development of the melanocyte
lineage, it must also regulate genes other than those involved in
melanogenesis as these genes are not essential for melanocyte development.
Hence, the unanswered question is whether Mitf is a crucial downstream factor
or a master regulator of melanocyte commitment and differentiation. One way of
addressing this question is to study the effect of Mitf expression on
undifferentiated cells.

In this study, we have transiently transfected fish ES-like cell lines with
the melanocyte-specific isoform of Mitf. This led to the induction of
cells that, by cell shape, pigmentation, melanosome motility and gene
expression pattern, were confirmed to be fully differentiated pigment cells.
This indicates that Mitf expression is sufficient for the proper
differentiation of medaka pluripotent stem cells into melanocytes.

Materials and methods

Cell culture and transfection

MES-1 and MES-2 cell lines were cultured in ESM 4 medium as previously
described (Hong et al., 1998).
The ES-like state of the cell lines was confirmed by transplantation to host
embryos and their ability to form chimaeras, most importantly the development
of donor-derived melanocytes in albino host
(Hong et al., 1998). Cells
were transfected with an expression vector containing the M isoform of the
Mitf cDNA from Xiphophorus (Delfgaauw et
al., 2003) under the control of the CMV promoter (pRc/CMV,
Invitrogen). The cells were cotransfected with pCMVgfp
(Hong et al., 1998), at a
ratio of 10 μg DNA of the Mitf expression vector/1 μg DNA of pCMVgfp.
After trypsinisation, the cells were resuspended in 400 μl of PBS,
electroporated in an Easyject electroporator at 250 V and 600 μF, and
seeded at 60% of confluence. As a control, MES cells were transfected with a
vector containing the cDNA of M-Mitf with a point mutation that introduces a
premature stop codon (Delfgaauw et al.,
2003). Experiments were carried out with the MES-1 and MES-2 cell
lines, with similar results. Transfection efficiencies were estimated counting
the number of GFP-expressing cells 24 hours after the transfection. The values
were between 10% and 15%. OLF, A2 and PSM cells were used as controls. OLF is
a medaka fibroblast-like cell line (Etoh
et al., 1988). A2 is an embryonic epithelial cell line
(Kuhn et al., 1979) and PSM is
a melanoma cell line (Wakamatsu,
1981) both derived from fish of the closely related genus
Xiphophorus. They were cultured as previously described
(Etoh et al., 1988;
Baudler et al., 1997) and
transfected by electroporation as the MES cell lines.

The number of melanocytes observed in a defined area of the culture dishes
(2 cm2) was counted at different days after the transfection. Three
replicates were counted from four different experiments. Differences were
lower than 15%.

For the motility assays 0.1 mM of norepinephrine (NE hydrochloride, Sigma)
was added to the medium (Uchida-Oka and
Sugimoto, 2001) of transfected MES cells. Cells were later
observed under the microscope to detect motile activity in the
melanosomes.

Preparation of RNA and RT-PCR

Transfected cells were harvested at different time points. Seeding of the
cells after the electroporation was considered time zero. Total RNA was
isolated with the Trizol reagent (GibcoBRL) from tissues or cell cultures and
2 μg were used for reverse-transcription using random primers. PCR was run
for various genes using the primers and the conditions described in
Table 1. One tenth of the
single strand cDNA products was used for each PCR amplification. Primers were
derived from medaka sequences available from public data bases
(Table 1). XmaMitf
primers were derived from the Xiphophorus sequence and were specific for the
transgene, no amplification was obtained with medaka skin cDNA (data not
shown). OlaMitf primers are specific for the M-isoform of medaka
Mitf. They were designed from genomic sequence reads of the medaka whole
genome shotgun sequence project available at
http://shigen.lab.nig.ac.jp/medaka/genome/indexen.html
Medaka actin was used for calibrating RNA amounts. Several independent
transfections were carried out with no variation in the expression results.
Every PCR was repeated at least four times.

Results

MES-1 and MES-2 cells (Fig.
1A,B) were co-transfected with plasmid expression vectors for the
Xiphophorus M isoform of Mitf and for GFP. A mutant Mitf did not induce any
phenotypic change in MES cells compared with nontransfected controls. However,
after transfection of the M-Mitf isoform, the cells reproducibly
started to adopt a melanocytic morphology (larger size and dendritic shape)
and to produce melanin. First, from day three to five, the differentiating
cells showed only some dendrites and were scarcely pigmented
(Fig. 1C,D), later they
presented more dendrites, were strongly pigmented and larger in size
(Fig. 1E).

Only GFP expressing transfected cells underwent the transition to pigment
cell morphology (Fig. 1F).
Thus, it can be excluded that a global nonspecific differentiation process was
taking place. The non-transfected cells retained their typical ES-like
morphology: small in size and round or polygonal in shape. Therefore it is
apparent that Mitf is the inducer responsible of the differentiation process
observed.

The number of melanocytes in the transfected cultures increased over time
(Fig. 1G) until it reached a
plateau at day ten (Fig. 2). At
this time, the expression of the transgene decreased, probably owing to loss
of the transfected DNA. This indicates that, as expected, the differentiation
process is cell autonomous and that the differentiated cells stop dividing.
The percentage of cells that differentiated represented between 30% and 50% of
the transfected cells, as determined 24 hours after transfection. The
transfected non-differentiated cells retained their ES cell-like morphology
and continued growing and dividing as the non-transfected cells. Therefore,
the ES cells tended to overgrow the differentiated cells at the end of the
3-week observation period.

Number of melanocytes observed in a defined area of the culture dishes
after transfection of the different cell lines with XmaMitf. Absolute
numbers are given because non transfected cells continue proliferating as
usual, thus a percentage value of melanocytes does not really reflect the
increase in the number of differentiated cells observed over time.

Transfection of Mitf into melanoma cells (PSM), or embryonic
epithelial cells (A2) did not lead to any morphological change
(Fig. 2). When Mitf
was introduced into a fibroblast cell line (OLF) a minimal effect was seen. At
day 10, a few pigmented cells appeared
(Fig. 1H), representing∼
0.1% of the transfected cells.

In lower vertebrates, melanocytes differentiate further to a stage that is
most prominently characterised by the ability of the melanosomes to be
translocated from the periphery of the cells to the center, thus giving the
cells a lighter or darker appearance. This process is mediated by a special
architecture of the cytoskeleton and is regulated by external physiological
signals, e.g. norepinephrin (NE), light, etc. With this response, the pigment
cells mediate the colour change of fish and amphibia. Hence, for a functional
assay the transfected MES cells were treated with NE in order to check the
ability of the melanocytes to aggregate the pigment granules. The effect was
clearly observed already within 30 minutes after adding the drug,
(Fig. 3A,B). Two hours later,
in many cells the pigment organelles were completely aggregated and retracted
to the perinuclear compartment (Fig.
3C). The same result was obtained by strong illumination of the
melanocytes (data not shown). This indicates that the differentiated cells
were fully functional pigment cells able to respond to physiological signals
coming from their environment.

For a molecular characterisation of the differentiation process, the
expression of various genes was tested by RT-PCR. At first, known target genes
of Mitf were analysed including the tyrosinase gene family: tyrosinase,
tyrosinase related protein 1 (Tyrp1) and dopachrome tautomerase (Dct or
Tyrp2), which are the enzymes involved in melanin synthesis. A clear induction
of the three genes was observed (Fig.
4A). No induction was detected in the cells transfected with the
mutant Mitf (Fig. 4B).
Concerning the timecourse of the induction, an increase in the expression
level of the three genes was detected over time, with Dct being the
earliest marker. Expression of this gene was already detected 2 hours after
transfection. Tyrp1 was the last of the three genes to be expressed,
being barely detectable at 8 hours and clearly visible at 24 hours. In
contrast to Tyrp1 or Dct, Tyrosinase expression decreased at 10 days. This is
also observed for the transfected Mitf and could be due to the loss of the
transgene.

To analyse whether there is any effect of the transgene on regulation of
the endogenous gene, the expression of the medaka Mitf gene was
determined at all time points. There was a low level of OlaMitf
expression in the non-transfected cells, which did not change after
introduction of the Mitf transgene at any time
(Fig. 4A).

Four genes were tested that are known markers of melanocyte
differentiation. Kit, a type III receptor tyrosine kinase (RTK) expressed in
melanocytes and melanoblasts, is essential for the development of neural
crest-derived melanocytes. In mammals, it has also an important role in
haematopoiesis and germ cell development
(Parichy et al., 1999;
Hou et al., 2000). Fms,
another type III RTK, is a closely related RTK to Kit. In mouse, it is
essential for the development of macrophage and osteoclast lineages; in
zebrafish it is required for pigment pattern development
(Parichy et al., 2000;
Parichy and Turner, 2003).
Aim1 is a putative integral melanosomal membrane protein that probably
functions in transporting certain substances required for melanin biosynthesis
(Fukamachi et al., 2001). The
endothelin receptor b (Ednrb) is important for the proliferation and
differentiation of neural crest derived cells, including melanocytes
(Opdecamp et al., 1998). For
Kit, Fms and Aim1, a clear induction of expression in the transfected cells
was observed (Fig. 4A). Fms is
the earliest appearing marker and Aim1 is the last
(Fig. 4A). The pattern of
Ednrb1 was different. This gene is expressed at low levels already in
the non-transfected cells. Two hours after transfection there was a clear
induction of this gene. By 4 hours, the expression was downregulated
again.

As an early differentiation marker Tbx2 was tested. This gene is a
member of the T-box transcription factor family that is expressed in
melanoblasts and melanocytes and is a known target of Mitf in mouse
(Carreira et al., 1998;
Carreira et al., 2000). It was
clearly induced (Fig. 4A) as
early as 4 hours after Mitf transfection.

As a general marker of cell differentiation, P53 expression was tested. P53
is a phosphoprotein that plays a general role in controlling cell
proliferation, growth arrest and apoptosis. It acts as a tumour suppressor
gene and has also been implicated in regulating cellular differentiation
(Sabapathy et al., 1997).
Induced expression of P53 was observed 2 hours after the transfection
(Fig. 4A).

In order to exclude the possibility that an unspecific upregulation of gene
expression is taking place in the Mitf expressing cells, the expression of
Myf5, a bHLH transcription factor that plays a key role in the skeletal muscle
lineage, was tested. No induction of this gene was observed
(Fig. 4A).

Melanocytes progress in their development in vivo through a precursor
stage, the neural crest cell, which can also differentiate to peripheral
neurons and glia - the Schwann cell cells. Occasionally, two or three
neuron-like cells per culture dish were observed after Mitf expression. Hence,
the expression of two pan-neuronal markers, NCAM and neuron-specific enolase
was assayed. Both genes were not detected in any of the Mitf or mutant Mitf
transfected cell lines (Fig.
4A,B).

The intimate relationship between melanocytes and glial lineages suggested
by the ability of Schwann cells to dedifferentiate to generate melanocyte
progenitors (Dupin et al.,
2003) suggested the possibility that some Schwann cell
differentiation might occur in our cultures. Therefore a typical Schwann cell
marker, Erbb3, which is the receptor for the growth factor neuregulin
(Garratt et al., 2000) was
tested. This gene is not expressed in the ES-like cells and was not induced in
Mitf-transfected MES during differentiation
(Fig. 4A). Sox10 is a
transcription factor required for the development of neurons, glia and pigment
cells. There was a transient expression at 24 hours for this gene, which did
not occur in the control transfections
(Fig. 4A,B). Sox10 is expressed
in differentiated Schwann cells (Jessen
and Mirsky, 2002); however, it is rapidly downregulated in
differentiating melanophores (Dutton et
al., 2001). Obviously, if there is some differentiation of Schwann
cells, it is a minor fraction and below the detection limit.

The transient expression of Sox10, a typical neural crest gene, could be
also an indication that the Mitf transfected ES cells express some
neural crest cell markers. To address this, the expression of two other neural
crest markers was analysed. AP2 is a transcription factor that regulates cell
growth, differentiation and programmed cell death
(Hilger-Eversheim et al.,
2000). Snail2 is a transcription factor involved in the formation
of premigratory neural crest cells
(Locascio et al., 2002). In
the case of AP2 a clear transient induction of the expression was observed,
with a peak at 4 hours after the transfection
(Fig. 4A). However, this
induction did not coincide with the transient expression of Sox10 at 24 hours.
For Snail2 the non-transfected cells already expressed this gene and
no change was observed after the transfection
(Fig. 4A). All genes tested as
markers for the Mitf induced differentiation were also analyzed in MES cells
transfected with the mutant Mitf (Fig.
4B). No changes of expression were detected.

All genes were also tested in the XmaMitf transfected fibroblasts,
where only in the case of tyrosinase, from 72 hours onwards a slight induction
could be detected (Fig. 5).
Dct, Ednrb, P53 and Snail2 were already expressed in the nontransfected cells.
No change in expression was observed for Dct and Snail2
(Fig. 5). In the case of Ednrb,
a slight increase followed by a strong decrease below the initial level was
detected (Fig. 5). An increase
in the expression level of P53 was observed from 24 hours on
(Fig. 5). For the rest of the
genes, expression was not detected at any time point. Thus, the effect of Mitf
expression on the OLF cells seems to be much more limited than on the
embryonic stem cells.

Expression assays of OLF cells after transfection with XmaMitf.
Controls are as for the MES cells (see Fig.
4).

Taken together, these results indicate that Mitf expression is sufficient
to initiate the complete differentiation from totipotent ES cells into the
melanocyte lineage, yielding terminally functional differentiated cells
expressing the specific genes of this cell type.

Discussion

The importance of Mitf as a determining factor of melanocyte development
was underlined when it was shown that expression of Mitf in mouse fibroblasts
resulted in the adoption of some melanocyte specific traits
(Tachibana et al., 1996).
Similarly, overexpression of zebrafish Mitf resulted in ectopic melanised
cells (Lister et al., 1999).
Thus, it appeared that, in at least some cells, the expression of Mitf was
sufficient to induce melanocyte-specific gene expression. In the present
study, the starting point were pluripotent embryonic stem cells. We obtained
completely morphologically differentiated melanocytes expressing all the
specific markers tested: the melanogenic enzymes, a melanosomal structural
protein, specific receptors and genes involved in the specification,
commitment and survival of this cell type. These results indicate that Mitf
can act as a master regulator of the melanocyte lineage, in that it is
sufficient to direct differentiation of embryonic stem cells into melanocytes.
The only other case so far reported of the transfection of a gene into ES
cells and subsequent induction of a specific lineage is the case of GATA
factors that directed mouse ES cells into the extra-embryonic endoderm lineage
(Fujikura et al., 2002). In that case, the ES-like cells did not reach a
terminal differentiation state indicating that these factors are responsible
only for the first step of the differentiation process. By contrast, Mitf
expression was sufficient to induce and complete the entire differentiation
process.

There was no induction of the endogenous Mitf gene in a kind of
autoregulatory loop. This is consistent with the need for upstream factors in
vivo that switch on Mitf at the right time during differentiation of
neural crest cells towards melanocytes
(Goding, 2000).

When Mitf was expressed in mouse fibroblasts
(Tachibana et al., 1996) after
antibiotic selection, only 2% of the transfected cells exhibited a
melanocyte-like appearance: dendritic shape and melanosome-like structures but
not melanin, explained by the finding that these contained a mutant
tyrosinase gene. In that experiment, cells that had already
differentiated were used, which are comparable to the OLF cells used in this
study, where our results are similar. However, the fact that the morphology of
the pigmented cells was not dendritic and that most of the specific markers
were not induced, raises doubts if it is acceptable to consider the observed
pigmented cells as true melanocytes or just as pigmented fibroblasts. The
differences between both cell lines in terms of `competence status' are
probably responsible for the differences in the effect of Mitf expression.
Only in the case of the ES-like cells does a true differentiation process seem
to happen.

In other studies on melanocyte differentiation, treatment with retinoic
acid (Watabe et al., 2002), or
seeding ES cells, melanocyte precursors or neural crest cells on a cell layer
(Yamane et al., 1999) have
been used as the inducing stimulus. These experiments led to melanocytes, but
the mechanisms that triggered the differentiation process could not be
elucidated. In addition, these experiments generated multiple cell lineages as
well as melanocytes. In our case, the simple initiation of Mitf expression
gives rise to terminally differentiated melanocytes from completely
undifferentiated ES-like cells. Only rarely were very few neuron-shaped cells
detected in the cultures. The absence of expression of the pan-neuronal
markers NCAM and neuron-specific enolase indicates either that they were not
completely differentiated or that they constitute only a negligible proportion
of the whole cell population; no other cell types were observed at the
microscopic level. We cannot, however, exclude the possibility that the
transient induction of Sox10 expression is a sign of some early
differentiation into the Schwann cell lineage.

A high percentage of the MES cells became melanocytes after Mitf
expression. However, not every transfected cell developed into a melanocyte.
This may be explained by the transient transfection experiment where usually
transgene DNA amounts vary in different cells. It appears reasonable to assume
that a threshold level of Mitf is necessary (J.B. and M.S., unpublished) to
trigger the differentiation process.

Cellular motile activities of fish pigment cells underlie colour change and
constitute crucially important strategies to avoid attack, obtain prey or for
communication with conspecifics (Fujii,
2000). In our study, this ability of the differentiated cells to
aggregate the pigment granules was used as a functional assay because it
requires a whole signal transduction and cytoskeleton motility machinery
(Uchida-Oka and Sugimoto,
2001). The clear response of the melanocytes to the treatment
confirms their status as terminally differentiated and functional
melanocytes.

Besides showing a typical melanocyte morphology, heavy pigmentation and
ability to aggregate the melanosomes, it was possible to analyse at the
molecular level for changes in the expression profile. Genes were analysed
that are known targets of Mitf as well as other melanocyte markers not
directly linked to Mitf so far.

In the case of the tyrosinase gene family, it was already clear that Mitf
binds and activates their promoters. The temporal pattern identifies
Dct as the earliest induced gene. This is comparable to the in vivo
situation in mouse embryos, where Dct is expressed early in the
melanoblasts, whereas tyrosinase and Tyrp1 are detected later
(Steel et al., 1992). In
zebrafish embryos, dct is also already expressed in melanoblasts
(Kelsh et al., 2000). In
humans, the level of activation of the TYRP1 promoter is lower than
that of the tyrosinase promoter (Fang et
al., 2002). This may also be true in medaka and could explain why
we can detect tyrosinase expression but not that of Tyrp1 in the OLF
cells. The presence of Dct even in the nontransfected fibroblasts is
surprising but also in non-transfected mammalian fibroblasts (NIH/3T3 cells)
Dct expression was reported
(Tachibana et al., 1996). It
is possible that this gene has a second, still unknown, function in
fibroblasts.

Concerning the melanocyte-specific markers, it has been reported that in
mammals Aim1 is transcriptionally modulated by Mitf, although the
mechanism of interaction is not yet clear
(Du and Fisher, 2002). In the
Mitf transfected MES cells, Aim1 is induced as a rather late
event. As Aim1 is not induced in the Mitf transfected OLF
cells, the interaction between Mitf and Aim1 may require factor(s)
which are not present in the OLF cells. For Mitf and Kit, complex interactions
were shown: Mitf is needed for the maintenance of Kit expression in
melanoblasts and Kit signalling modulates Mitf activity and stability in
melanocyte cell lines (Hou et al.,
2000). There is an E-box motif in the Kit promoter
through which Mitf can activate Kit
(Tsujimura et al., 1996).
Thus, the observation of Kit upregulation in Mitf expressing MES
cells may be due to a direct interaction of Mitf with the Kit
promoter. Again, the lack of induction in the fibroblast cells suggests
special requirements of this interaction. A connection between Mitf and Fms
had been established in mammals, where Fms seems to act in the osteoclast
lineage like Kit in the melanocyte lineage
(Kawaguchi and Noda, 2000). In
zebrafish an unexpected role has been described for Fms in pigment pattern
development where Kit and Fms are required by two separate populations of
pigment cells (Parichy et al.,
2000; Parichy and Turner,
2003). At present, we cannot say if in MES cells Kit and Fms are
expressed by different cells or simultaneously.

For Ednrb, a transient activation was reproducibly observed. However, the
possible presence of two Ednrb orthologues in medaka (alike the
situation in chicken and zebrafish) makes it difficult to infer the
implication of this expression. Ednrb is expressed in neural crest cells
before the onset of Mitf, suggesting that in these experiments Ednrb
may be considered as a neural crest stage marker of the differentiation
process rather than a melanocyte-specific gene. It has been proposed that
Ednrb functions in neural crest suppressing the differentiation of early stage
melanocytes and enteric neuron precursors, rendering them competent for
migration and accessible to mitogenic influence of other factors
(McCallion and Chakravarti,
2001; Shin et al.,
1999). Thus, when terminal differentiation is initiated, it may be
necessary to repress Ednrb expression. In our experiments Ednrb
expression returned to basal levels very rapidly, before expression of
tyrosinase and Trp1 started and any morphological signs of
pigment cell differentiation was observed. Intriguingly, this transient
activation followed by a downregulation was seen in both MES and OLF cell
lines, after Mitf transfection.

The results obtained with Tbx2 confirm a link with Mitf in medaka. This
family of transcription factors is involved in the maintenance of cell
identity. Tbx2 is a known target of Mitf in mouse and it has been
proposed that it may be the initiation of one of the pathways that Mitf uses
to play its role in melanocyte development and survival
(Carreira et al., 1998;
Carreira et al., 2000).

Mouse ES cells express high levels of P53 and differentiation results in a
reduction of the P53 levels (Sabapathy et
al., 1997). By contrast, in medaka, P53 expression increases
during development. In MES cells, expression is not detected
(Chen et al., 2001). Our
results agree with these previous data confirming P53 as a general marker of
embryonic cell differentiation in medaka. Expression of P53 is also induced
when the adult fibroblasts (OLF) are transfected, suggesting a link between
Mitf and P53 that is still unclear. We observed an erratic expression of some
genes that are expressed in neural crest cells in vivo. However, this erratic
expression did not occur at exactly the same time. Ednrb was induced
already at 2 hours, AP2 at 4 hours and Sox10 at 24 hours.
Although we do not know the in vivo time course of gene expression in medaka
neural crest cells, the expression profile of these three genes in the
Mitf transfected ES cells is difficult to explain. For Sox10 it is
clear that in vivo it is required for Mitf expression
(Dutton et al., 2001;
Elworthy et al., 2003), but as
yet there is no evidence for the opposite. The induction of Sox10 was
seen after some melanocyte specific genes already started to express. Sox10 is
also expressed in some melanocyte cell lines
(Goding, 2000). The expression
of the three genes might therefore be due to the transient presence of other
cell types or their precursor stages. Alternatively, the expression may be due
to the in vitro conditions. Another hypothesis could be that the ES cells on
their enforced path to become melanocytes recapitulate the expression of some
genes which in vivo are characteristic for the neural crest cell stage. Of
course, a `pure' neural crest-step cannot be expected as Mitf is downstream of
the neural crest induction and is expressed only in a part of the neural crest
derivatives. Our data do not allow us to define the mechanism that make the ES
cells express these neural crest markers after Mitf expression. More
experiments are necessary to clarify this point and methods have to be
developed that allow the analysis of expression on the single cell level in
the differentiating ES cells.

The wide use of Snail2 as a neural crest marker and the recent study which
strongly suggest that Mitf interacts with Slug (the closest Snail2 homolog in
mammals) (Sánchez-Martín et
al., 2002), prompted us to look for the effect of Mitf expression
on this gene. But surprisingly, Snail2 is already present in the
non-transfected MES and OLF cells, and its expression is not affected by Mitf.
In zebrafish embryos, Snail2 is expressed in gastrula stages; later, its
expression is suppressed in some lineages but not in neural crest and its
derivatives (Thisse et al.,
1995). It has also been shown that Snail2 controls the expression
of genes involved in cell properties affecting the interactions of neighboring
cells, such as cell-cell adhesion (Thisse
et al., 1995). Such interactions are probably very important in
vitro and may explain its expression in both cell lines. Therefore Snail2
cannot be used in our differentiation system as a marker for neural crest as
it is used in vivo. More experiments need to be carried out in order to
clarify the role of this factor in these cell lines.

Mitf is the earliest known marker of commitment to the melanocyte lineage
and most of the signalling molecules or transcription factors implicated
genetically in melanocyte development affect either Mitf expression or its
function (reviewed by Goding,
2000). Our data support a proposed role for Mitf as a master
developmental regulator, the expression of which is sufficient to initiate and
complete the differentiation of medaka pluripotent cells into melanocytes.

Many questions remain to be addressed, among them the role of the genes
upstream of Mitf, the ability of other Mitf isoforms to direct
differentiation of MES cells, and the molecular mechanisms of Mitf interaction
with its target genes, direct or indirect. The fact that MES cells are
embryonic stem cells and not embryonic carcinoma cell lines, and that the
formation of embryoid bodies is not necessary after the transfection, makes
the medaka ES cell system an elegant model that will help to achieve more
insight into this and other differentiation pathways.

Acknowledgments

We thank J. Delfgaauw and J. Altschmied for providing the Mitf expression
vectors; Ute Hornung, Toni Wagner and Mariko Kondo for their help with the
revised version of the manuscript; and C. Winkler for critical reading and
discussion. We are grateful to the Medaka Genome Initiative and the Medaka
Whole Genome Shotgun Sequencing Consortium for making their data available.
This work was supported by grants to M.S. from the Deutsche
Forschungsgemeinschaft (SFB 465) and Fonds der Chemischen Industrie.

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melanocytes, but is not essential for hematopoiesis or primordial germ cell
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